There are about 100,000 different proteins expressed in eukaryotic systems. Protein structural models are a unique source of information: location and properties of binding sites in toxins; domain structure of lipoproteins; molecular contact and recognition. Generating correct and detailed structural models of proteins is aided by the ability to obtain and analyze a protein in its crystal form.
Proteins are macromolecules (heteropolymers) made up from 20 different amino acids, also referred to as residues. For proteins below about 40 residues the term peptide is frequently used. A certain number of residues is necessary to perform a particular biochemical function, and around 40-50 residues appears to be the lower limit for a functional domain size. Protein sizes range from this lower limit to several hundred residues in multi-functional proteins.
Proteins can be several hundred residues long and fold into a 3-dimensional structure. It is therefore quite understandable that protein molecules have irregular shapes and are not ideally suited to be stacked into a periodic lattice, i.e., a crystal. Protein crystals are thus very fragile, soft and sensitive to environmental variations. Protein crystals contain on average 50% solvent, mostly in large channels between the stacked molecules of the crystal. The interactions holding the molecules together are usually weak, hydrogen bonds, salt bridges, and hydrophobic interactions.
The structures of many important proteins remain a mystery simply because researchers are unable to obtain crystals of high enough quality or large enough size. Generally, for useful measurements to be obtained, crystals must have dimensions of approximately 0.3 mm to 1.0 mm, and the protein molecules must be arranged in an orderly, repeating pattern.
In order to obtain a crystal, the protein molecules must assemble into a periodic lattice. To bring the protein molecules into close association so that nucleation may occur, one typically starts with a solution with a high protein concentration (2-50 mg/ml) and adds reagents that reduce the solubility close to spontaneous precipitation. By slow further concentration, and under conditions suitable for the formation of a few nucleation sites, small crystals may start to grow. Often many conditions have to be tried to succeed. This is usually done by initial screening, followed by a systematic optimization of conditions. Crystals should to be sub-mm range in each direction to be useful for conventional diffraction experiments.
Other techniques for growing protein crystals, such as ‘sitting drops’, ‘dialysis buttons’, and ‘gel and micro batch’ techniques are known in the art. Devices for promoting crystallization include the hanging-drop, sitting-drop, sandwich-drop, dialysis, micro batch or microtube batch devices (U.S. Pat. Nos. 4,886,646, 5,096,676, 5,130,105, 5,221,410 and 5,400,741; Pav et al., Proteins: Structure, Function, and Genetics, 20, pp. 98-102 (1994); Chayen, Acta. Cryst., D54, pp. 8-15 (1998), Chayen, Structure, 5, pp. 1269-1274 (1997), D'Arcy et al., J. Cryst. Growth, 168, pp. 175-180 (1996) and Chayen, J. Appl. Cryst., 30, pp. 198-202 (1997), incorporated herein by reference). Microseeding may be used to increase the size and quality of crystals.
In iterative drug design, crystals of a series of protein or protein complexes are obtained and then the three-dimensional structure of each crystal is solved. Such an approach provides insight into the association between the proteins and compounds of each complex.
Notwithstanding the variety of methods practiced, what are constantly sought are faster, less expensive methods of crystallizing biomolecules, and, in particular, proteins.
Nucleation requires higher levels of saturation than those associated with metastable phases amenable to crystal growth. An environment that favors a higher local concentration of macromolecules may lower the energy barrier for nucleation. Compositionally modulated superlattices have been identified which act as potent and highly specific catalysts for the nucleation of many different protein crystals. (See “Nanoengineered Surfaces for the Epitaxial Nucleation of Protein Crystals”, Robert Haushalter and Ted X. Sun, Parallel Synthesis Technologies and Alexander McPherson, Univ. of California, Irvine, Calif.).
What is needed is a means of providing an environment that favors a high local concentration of the macromolecules of interest, and thereby fostering nucleation and subsequent crystallization. What is needed are ways to bring protein molecules or residues in close association under conditions so as to promote the weak bonding necessary for protein crystallization. Further, what is needed is a rapid means of calibrating conditions for a high through-put protein purification, nucleation and/or crystal growth.
What is also needed is a “bottoms up” nanoscale means to promote protein-protein bonding and crystal growth. What is also needed is a controllable nanoscale environment to assemble biomolecules, including proteins, in periodic lattice formations. What is further needed is the ability to grow crystals suitable for diffraction analysis on a programmable nanoscale array.
What is also desired is a method for creating a surface designed for seeding organic crystals and especially protein crystals. Further desired is a means to refine and purify proteins in mixtures and solutions. What is needed are purifying devices and methods that facilitate protein nucleation, protein crystallization and other protein identification and testing steps, as well as refining or purifying protein-based drugs or drug-precursors.